Small regulatory RNAs: from bench to bedside – a keystone symposia meeting report

ABSTRACT The Keystone Symposium ‘Small Regulatory RNAs: From Bench to Bedside’ was held in Santa Fe, New Mexico from May 1–4, 2022. The symposium was organized by Frank J. Slack, Jörg Vogel, Ivan Martinez and Karyn Schmidt, and brought together scientists working in noncoding RNA biology, therapeutics, and technologies to address mechanistic questions about small regulatory RNAs and facilitate translation of these findings into clinical applications. The conference addressed four specific aims: Aim 1. Focus on the exciting biology of small regulatory RNAs, highlighting the best current research into the role that small RNAs play in fundamental biological processes; Aim 2. Focus on the latest efforts to harness the power of these RNAs as agents in the fight against disease and provide the basic understanding that will drive the invention of powerful clinical tools; Aim 3. Attract leaders from both academia and industry working in small RNAs to one place for critical discussions that will advance the field and accelerate the bench to bedside use of this technology; Aim 4. Provide a stimulating environment where students, postdoctoral researchers and junior investigators, along with scientists from Biotechnology and Pharmaceutical companies specializing in small regulatory RNAs, can present and discuss their research with the best minds in the field.


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The Keynote address entitled 'Viral Noncoding RNAs: New Insights into RNA Structure' was given by Joan A. Steitz from Yale University. She talked about the Kaposi's sarcoma-associated herpesvirus (KSHV) noncoding, polyadenylated nuclear (PAN) RNA that is required for late protein synthesis and virus production [1][2][3][4][5]. Remarkably, PAN RNA accumulates to about half a million copies in the nucleus and accounts for about 80% of polyadenylated RNAs in the infected cell [1,4]. This high-level accumulation requires an RNA stabilization element called the 'element for nuclear expression' (ENE), which is located near the 3' end of PAN RNA [6][7][8], and protects this viral RNA from deadenylation and decay via sequestration of the poly(A) tail [7][8][9]. ENEs are also found in mRNAs as well as noncoding RNAs [10,11], and they counteract the negative effects of intron loss in transposon mRNAs [12]. She presented the high-resolution crystal structure of a double ENE (dENE) complexed with 28-mer poly(A) to reveal novel modes of interactions between the dENE and poly(A) tail [13]. These findings greatly expand our knowledge of poly(A) tail function in RNA biology.
The first session focused on the role that small RNAs play in cancer. Joshua T. Mendell (University of Texas Southwestern Medical Center) described different modes of microRNA (miRNA)-target interactions and specific post-transcriptional modifications that determine the fate of the target RNA and the Argonaute protein [14,15]. George A. Calin (University of Texas MD Anderson Cancer Center) provided insights into unconventional miRNA functions [16], ultraconservation in human cancers [17,18], and small molecules targeting oncogenic miRNAs [19]. Christine Eischen (Thomas Jefferson University) gave a talk on identifying and characterizing critical miRNAs that target multiple messenger RNAs in one or more cellular pathways in tumour development or suppression [20]. Aurora Esquela-Kerscher (Eastern Virginia Medical School) showed that miR-888 cluster members are promising clinical targets for aggressive prostate cancer [21,22].
The second session highlighted miRNAs in therapeutics. Ivan Martinez (Western Virginia University) showed that SARS-CoV -2 infection specifically reduces cytoplasmic Drosha expression and that alternative splicing of Drosha is regulated differently by distinct SARS-CoV-2 variants. Vishal Patel (University of Texas Southwestern Medical Center) highlighted the promises and challenges of developing anti-miR-17 therapy for autosomal dominant polycystic kidney disease [23]. Raman Bahal (University of Connecticut) gave a talk on targeting miR-21 and miR-10b for glioma therapy by using next generation gamma peptide nucleic acids (γPNAs) and polylactic acid-hyperbranched polyglycerol (PLA-HPG) [24][25][26]. Murugaiyan Gopal (Harvard Medical School) showed that T-cell intrinsic miR-92a inhibits Treg induction and promotes Th17 differentiation by targeting Foxo1 and that miR-92a silencing represents a promising approach to restore immune tolerance in MS patients by modulating Treg and Th17 responses [27].
The third session examined diverse small regulatory RNAs. Richard I. Gregory (Boston Children's Hospital/Harvard Medical School) showed that METTL1-mediated m 7 G modification of Arg-TCT tRNA drives oncogenic transformation and that targeting oncogenic tRNA Arg-TCT is a promising anti-cancer strategy [28]. Jörg Vogel (University of Würzburg) provided insights into the promises and challenges of using antisense peptide nucleic acids (PNAs) as speciesspecific programmable RNA antibiotics for selective targeting of individual bacterial species or genes [29]. Sandra L. Wolin (NCI, National Institutes of Health) showed that damaged tRNAs are signalling molecules that can bind to the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, leading to activation of an RNA-repair operon [30]. Gisela T. Storz (National Institutes of Health) gave a talk on small regulatory RNAs that are derived within coding sequences and how they function as base-pairing RNAs to regulate protein-coding sequences [31,32]. Daniel Cifuentes (Boston University) showed that the blood-specific miRNA miR-144 regulates chromatin condensation during erythrocyte maturation by down-regulating HMGN2 expression. Ruben Garcia Martin (Joslin Diabetes Center/Harvard Medical School) provided insights into defining cell-type specific miRNA codes controlling exosomal sorting and cellular retention [33].
The next session focused on small regulatory RNA functions and non-canonical pathways. Shobha Vasudevan (Massachusetts General Hospital) discussed the posttranscriptional regulatory mechanisms involving regulatory noncoding RNAs that contribute to chemoresistance and persistence of quiescent cancer cells. Martin J. Simard (CRCHU de Québec-Université Laval) described the function and regulation of the miRNA-mediated gene silencing pathway during animal development and showed that serine 642 dephosphorylation of Argonaute ALG-1 controls miRNA-induced silencing complex (miRISC) formation [34]. Monika Gullerova (University of Oxford, Wedham College) showed that Dicer dependent tRNA-derived small RNAs (tsRNAs) can mediate nascent RNA silencing in nuclei and discussed the therapeutic potential of novel synthetic tsRNAs-based agents [35]. Elena Piskounova (Weill Cornell Medicine) showed that selenocysteine tRNA methylation regulates stress resistance in melanoma metastasis and outlined the concept of reprogramming the metastatic proteome through tRNA wobble modifications. Soo Mi Lee in the Virology Program at Harvard Medical School, showed that cellular miR-127-3p suppresses KSHV-induced transformation and tumorigenesis via down-regulation of SKP2, identifying the miR-127-3p/SKP2 axis as a viable therapeutic avenue for Kaposi's sarcoma [36].
The next session examined therapeutic opportunities for small RNAs. Mofang Liu (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) showed that PIWI/ piwi-interacting RNAs (piRNAs) regulate protein-coding genes in Drosophila early embryos and mouse spermatids beyond transposon silencing, and that piRNA 3' ends are defined by the PIWI-Ins element and contribute to piRNA targeting efficiency [37]. Stefan Engelhardt (Technical University of Munich) provided an overview of miRNA functions in the cardiovascular system and the current state of miRNA-targeted therapeutics and delivery platforms for cardiovascular disease [38]. Karyn Schmidt (Alnylam Pharmaceuticals) gave an overview of Alnylam's RNAi therapeutics platform and how Alnylam is leveraging a better mechanistic understanding of the RNAi pathway to improve duration, potency, specificity, and delivery of RNAi therapeutics [39,40]. Frank J. Slack (BIDMC Cancer Center/Harvard Medical School) provided an overview of a co-clinical trial with cobomarsen (MRG-106), a miR-155 targeting oligonucleotide, performed with miRagen Therapeutics in patients with diffuse large B-cell lymphoma (DLBCL) and cutaneous T cell lymphoma (CTCL) [41], and described the potential for miRNAs as immune checkpoint inhibitors [42]. John T. Powers (Dell Medical School at the University of Texas at Austin) gave an overview of the mechanisms of let-7 disruption in neuroblastoma [43] and showed that multiple isoforms of let-7 agonist H19 long noncoding RNA (lncRNA) are highly expressed in MYCN non-amplified neuroblastoma.
The final session highlighted emerging technologies and novel applications for small RNAs. William J. Greenleaf (Stanford University) described a high-throughput sequencing-based method to identify rules for miRNA target binding and cleavage by Argonaute2 (AGO2) and showed that knockdown can be explained by a biophysical RNAi kinetic model incorporating binding and cleavage rates in live cells [44]. Sabrina R. Leslie (University of British Columbia) described a tether-free, quantitative, single-molecule imaging platform (Convex Lens-induced Confinement (CLiC) microscopy) [45] that enables visualization of RNA interactions under cell-like conditions to help understand and develop RNA therapeutics. loannis S. Vlachos (Beth Israel Deaconess Medical Center) gave an overview of the concept of cancer immunosurveillance and described how single-nucleotide polymorphisms in 3' UTRs can contribute to the cancer immunoediting process. Byunghee Yoo (Massachusetts General Hospital/Harvard Medical School) showed that RNAi-based PD-L1 inhibitor, MN-siPDL1, is efficacious in highly aggressive models of pancreatic ductal adenocarcinoma with intense desmoplasia [46].
At the end of the last session, Frank J. Slack, Jörg Vogel, Karyn Schmidt, and Ivan Martinez concluded the conference by sharing their thoughts on the growing importance of small noncoding RNAs in medical therapeutics, highlighting the cutting-edge basic and translational research presented at the meeting, with a focus on the inclusion of all kinds of small noncoding RNA species and the diverse range of model systems employed to study them. Importantly, a number of collaborations are already beginning to form -stemming from the conference presentations. Each of the organizers agreed that specific outcomes and promising future directions included: A highlight of the number of advances the field of small noncoding RNAs is currently experiencing; A focus on the connections and collaborations formed from this meeting, and the incorporation of novel techniques into laboratories based on the conference presentations; An emphasis on how the discoveries and therapeutics presented at this meeting are not necessarily islands, but that these discoveries could change how we think about biology and new medicines; A forward focus on what is achievable in the coming years through the discovery of new small RNA species and their implication for our understanding of disease processes and the consequent discovery of novel, RNA-based therapeutics.
The conference offered poster sessions and diverse workshops, which provided an opportunity for researchers to present on emerging topics in the small regulatory RNA field, as well as a career roundtable, which resulted in discussions, guidance and mentoring for early career professionals and facilitated excellent networking opportunities. The emerging topics workshop covered a wide range of topics, including the role of miRNAs in Islet Autoimmunity, miRNA-based antitumour therapy, and their role in mitochondrial diseases. The roundtable included sessions from Christine Eischen from Thomas Jefferson University, Stefan Engelhardt from the Technical University of Munich, and John T. Powers from Dell Medical School at the University of Texas at Austin. This represented three different research stages in academia, and they were able to answer specific questions from studentstailoring advice based on student experience and goals.
Overall, conference attendees experienced a set of presentations (from the podium and the posters) at the cutting edge of RNA therapeutics, with a growing appreciation for the potential and challenges that lie ahead. Given the optimal size of the conference, attendees had the opportunity for extensive interactions that will drive innovations in the future. The organizers concluded that the four aims of the conference were achieved.

Acknowledgments
This conference was supported by Cell Research.

Disclosure statement
NFJS was a scientific advisor for miRagen Therapeutics but has no financial conflicts. No potential conflict of interest was reported by the other authors.

Funding
The work was supported by the National Cancer Institute

Data availability statement
Data sharing is not applicable to this article as no new data were created or analysed in this study.